13426
Biochemistry 1996, 35, 13426-13433
Tubulin Conformation and Dynamics: A Red Edge Excitation Shift Study
Suranjana Guha,‡ Satinder S. Rawat,§ Amitabha Chattopadhyay,§ and Bhabatarak Bhattacharyya*,‡
Department of Biochemistry, Bose Institute, Centenary Building, Calcutta 700 054, India, and Centre for Cellular and
Molecular Biology, Uppal Road, Hyderabad 500 007, India
ReceiVed May 27, 1996; ReVised Manuscript ReceiVed August 6, 1996X
ABSTRACT: The fluorescence emission maximum of a polar fluorophore in viscous medium often shows
a dependence on excitation wavelength, a phenomenon which is named red edge excitation shift (REES).
We have found that the fluorescence spectra of the tubulin tryptophans exhibit a REES of about 7 nm.
Also, their steady state fluorescence polarization and mean lifetimes show a dependence on both excitation
and emission wavelengths. These results indicate that the average tryptophan environment in tubulin is
motionally restricted. Although the tryptophan(s) responsible for the observed REES effect could not be
localized, it could be concluded from energy transfer experiments with the tubulin-colchicine complex
that the tryptophan(s) participating in energy transfer with bound colchicine probably does not contribute
to the REES. A REES of 7 nm was also observed in the case of colchicine complexed with tubulin.
However, such a REES was not seen in similar studies with the B-ring analogs of colchicine, viz.
2-methoxy-5-(2′,3′,4′-trimethoxyphenyl)tropone (called AC because it lacks the B ring of colchicine) and
deacetamidocolchicine (which lacks the acetamido substituent at the C-7 position of the B ring). There
may be two possible reasons to explain these data. (1) Structural differences between colchicine and its
analogs may give rise to differences in their excited state dipole moments which will directly affect the
extent of REES, and (2) The B-ring substituent, hanging outside the colchicine binding site on the β-subunit
of the tubulin dimer, probably makes contact with the R-subunit of tubulin and imparts a rigidity to that
region of the protein, which facilitates the REES.
Fluorescence spectroscopy represents a sensitive approach
for investigating both structural and molecular dynamic
properties of systems of biological interest. The fluorescence
emission spectrum for most polar fluorophores in nonviscous
medium is generally observed to be independent of excitation
wavelength. However, when such a fluorophore is in a polar,
viscous environment, i.e., where its mobility is restricted,
the wavelength of maximum fluorescence emission frequently shows a dependence on excitation wavelength.
Excitation at the extreme red edge of the absorption spectrum
causes a red-shifted emission in these cases. This phenomenon is popularly known as the red edge excitation shift
(REES)1 (Chen, 1967; Galley & Purkey, 1970; Castelli &
Forster, 1973; Itoh & Azumi, 1975; Demchenko, 1982;
Lakowicz & Keating-Nakamoto, 1984; Mukherjee & Chattopadhyay, 1995).
For a polar fluorophore, there exists a statistical distribution
of solvation states based on their dipolar interactions with
the solvent molecules in both the ground and excited states.
A polar fluorophore, in the ground state, interacts with the
surrounding solvent dipoles to remain in an energetically
favorable orientation. Upon excitation, the dipole moment
of the fluorophore changes, and consequently, the solvent
* Author to whom correspondence should be addressed. B. Bhattacharyya, Department of Biochemistry, Bose Institute, Centenary
Building, P-1/12 C. I. T. Scheme VII M, Calcutta 700 054, India. Fax:
(91) (33) 334-3886. E-mail: [email protected].
‡ Bose Institute.
§
Centre for Cellular and Molecular Biology.
X Abstract published in AdVance ACS Abstracts, September 15, 1996.
1 Abbreviations: PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid);
EGTA, ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic
acid; GTP, guanosine triphosphate; REES, red edge excitation shift;
AC, 2-methoxy-5-(2′,3′,4′-trimethoxyphenyl)tropone; DAAC, deacetamidocolchicine.
S0006-2960(96)01251-2 CCC: $12.00
dipoles have to reorient themselves around the excited state
fluorophore in order to reattain the energy minima (solvent
relaxation). This reorientation process consists of two
components: (i) electron redistribution in the solvent molecules which occurs within 10-15 s and (ii) the physical
reorientation of the solvent molecules. This latter process
is slow and dependent on the viscosity of the medium. In a
nonviscous solvent, the solvent dipoles can reorient around
the excited state polar fluorophore within 10-12 s, i.e., within
its excited state lifetime (10-9 s). Hence, no matter what
the excitation wavelength is, emission always occurs from
the solvent-relaxed state. In a viscous solvent, solvent
relaxation is restricted and occurs on the time scale of 10-9
s or longer. This results in differential extents of solvent
reorientation around the excited state fluorophore, with each
excitation wavelength selectively exciting a different average
population of fluorophores. Thus, excitation at the red edge
of the absorption spectrum (by lower-energy quanta) selectively excites those fluorophores which interact more strongly
with solvent molecules in the excited state, i.e., those around
which solvent molecules are oriented in a way similar to
that found in the solvent-relaxed state. Emission from this
state will be red-shifted. On the other hand, excitation at a
wavelength central to the absorption band selects for those
fluorophores around which solvent relaxation has not yet
occured and a blue-shifted emission is observed. This, in
brief, is the principle underlying the REES phenomenon
(Macgregor & Weber, 1982; Lakowicz & Keating-Nakamoto, 1984; Demchenko, 1986, 1988a; Demchenko &
Ladokhin, 1988; Mukherjee & Chattopadhyay, 1995).
The origin of the REES thus lies in the change in
fluorophore-solvent interaction in the ground and excited
states, brought about by a change in dipole moment of the
© 1996 American Chemical Society
A REES Study of Tubulin
fluorophore upon excitation, and the rate at which solvent
molecules reorient around the excited state fluorophore. Since
the REES is observed only under conditions of restricted
mobility, it has been used as a potential tool to estimate the
fluorophore (both intrinsic and extrinsic) environment in
organized biological assemblies such as membranes, micelles,
and proteins. Various membrane phenomena have already
been studied by incorporating lipids labeled with fluorescent
reporter groups such as 7-nitrobenz-2-oxa-1,3-diazol-4-yl
(NBD) into model membranes (Chattopadhyay & Mukherjee,
1993) and micelles (S. S. Rawat, S. Mukherjee, and A.
Chattopadhyay, unpublished observations). The fluorescent
probe 6-(p-toluidinyl)-2-naphthalenesulfonic acid (TNS)
shows a REES when bound to proteins such as apomyoglobin, β-lactoglobulin, β-casein, and several serum albumins
(Demchenko, 1982; Lakowicz & Keating-Nakamoto, 1984;
Albani, 1992). A REES has also been reported in proteins
like mellitin (Chattopadhyay & Rukmini, 1993), gramicidin
(Mukherjee & Chattopadhyay, 1994), the intact eye lens (Rao
et al., 1989), and cytochrome b5 (Ladokhin et al., 1991). The
REES of tryptophan fluorescence is usually not observed in
proteins having either very short (307-323 nm) or very long
(341-350 nm) wavelength emission maximum, but it is
usually observed in proteins that emit at intermediate
wavelengths (Demchenko, 1988b). Our protein, tubulin, falls
in this latter category. There are eight tryptophan residues
per tubulin dimer, and on excitation at 280 nm, their emission
maximum is at 333 nm. It is thus of interest to examine
whether the REES takes place in tubulin. In this paper, we
report some observations upon red edge excitation of the
tubulin tryptophans. We have also done REES studies with
the antimitotic drug colchicine and its structural analogs when
bound to tubulin. These drugs have a single, specific binding
site on the protein, and our study provides useful information
about not only the dynamic properties of the region of the
protein containing the binding site but also the involvement
of different parts of the colchicine molecule in the binding
phenomenon.
Biochemistry, Vol. 35, No. 41, 1996 13427
water bath for maintaining constant temperature in the cell
holder. A 1 cm path length quartz cuvette was used for all
experiments. For experiments that monitor changes in
emission maxima with excitation wavelength, excitation and
emission slits with a band-pass of 5 nm were used. For
polarization measurements, excitation and emission slits
corresponding to band-passes of 5 and 10 nm, respectively,
were used. Polarization experiments were performed using
Hitachi polarization accessories. The fluorescence intensity
components (Ivv, Ivh, Ihv, and Ihh), in which the subscripts
refer to the horizontal (h) or vertical (v) positioning of the
excitation and emission polarizers, respectively, were used
to calculate steady state fluorescence polarization using the
following equation (Chen & Bowman, 1965)
P)
(1)
where G is the grating correction factor and is equal to Ihv/
Ihh. In all cases, background intensities of the buffer were
subtracted from the sample spectra to eliminate errors due
to scattering artifacts.
Fluorescence lifetimes were calculated from time-resolved
decay of fluorescence intensity using a Photon Technology
International LS-100 luminescence spectrophotometer operated in the time-correlated, single-photon-counting mode. A
thyraton-gated nanosecond flash lamp filled with nitrogen
(16 ( 1 in. of mercury vacuum) was used and the machine
run at 22-25 kHz. Lamp profiles were measured at the
excitation wavelength using Ludox as the scatterer. All
experiments were performed using slit widths of 6 nm or
less. To optimize the signal to noise ratio, 5000 counts were
collected in the peak channel. The sample and scatterer were
alternated after 10% acquisition to minimize errors due to
shape and timing drifts during data collection. The data
obtained were analyzed on an IBM PC/AT computer, and
the intensity decay curves were fitted as a sum of exponential
terms:
F(t) ) Ri exp(-t/τi)
MATERIALS AND METHODS
Materials. PIPES, EGTA, GTP, and colchicine were
obtained from Sigma Chemical Co. Urea was from Aldrich
Chemical Co., and deacetylcolchicine from Molecular Probes.
Other colchicine analogs used in this study were gifts from
T. J. Fitzgerald, Florida A & M University, Tallahassee, FL.
All other reagents were of analytical grade.
Purification of Tubulin. Tubulin was isolated from goat
brains by two cycles of GTP and temperature dependent
assembly and disassembly in buffer containing 50 mM
PIPES, 1 mM EGTA, and 0.5 mM MgCl2 (pH 7), followed
by two further cycles in 1 M glutamate buffer (Hamel &
Lin, 1981). The purified tubulin, freed from microtubuleassociated proteins, was stored in aliquots at -70 °C. Protein
concentrations were estimated by the method of Lowry et
al. (1951).
Complexes of colchicine with tubulin were prepared by
incubating the protein (5 µM) and drug (10 µM) at 37 °C
for 1 h. All experiments were done in buffer containing 20
mM sodium phosphate, 1 mM EGTA, and 0.5 mM MgCl2
at pH 7.0 and 25 °C, unless otherwise mentioned.
Fluorescence Spectroscopic Studies. Steady state fluorescence measurements were done on a Hitachi F-3000
fluorescence spectrophotometer, fitted with a circulating
Ivv - GIvh
Ivv + GIvh
(2)
where Ri is the preexponential factor representing the
fractional contribution to the time-resolved decay by the
component having a lifetime of τi. The decay parameters
were obtained using a nonlinear least-squares, iterative
program based on the Marquardt algorithm (Bevington,
1969). The goodness of each fit was checked from the chisquare values, the weighted residuals (Lampert et al., 1983),
and the autocorrelated function of the weighted residuals
(Grinvald & Steinberg, 1974). A fit was considered acceptable when plots of the weighted residuals and the autocorrelation function showed random deviation about zero with
a chi-square value generally not exceeding 1.5. Mean
lifetimes (τ) for biexponential decays of fluorescence were
then calculated using the equation (Lakowicz, 1983)
τ)
R1τ12 + R2τ22
R1τ1 + R2τ2
(3)
RESULTS
Red Edge Excitation of the Tubulin Tryptophans. Figure
1A shows the fluorescence emission spectra of the tubulin
tryptophans upon excitation at different wavelengths. As
13428 Biochemistry, Vol. 35, No. 41, 1996
FIGURE 1: (A) Fluorescence emission spectra of tubulin, at 25 °C,
in buffer containing 20 mM sodium phosphate, 1 mM EGTA, and
0.5 mM MgCl2 at pH 7.0 at different excitation wavelengths. The
excitation wavelengths were 280 nm (s), 290 nm (- - -), and 300
nm (- · -). All spectra were normalized at the emission maximum,
and the tubulin concentration was 2 µM. (B) Fluorescence emission
maximum of 2 µM tubulin plotted as a function of excitation
wavelength in the absence (b) and presence (O) of 8 M urea, at 25
°C. (C) Changes in the wavelength of maximum emission of 2
µM tubulin upon red edge excitation at different temperatures: 15
°C (4), 25 °C (0), and 37 °C (9).
seen from the figure, the emission spectra of the tryptophans
in tubulin show considerable dependence on excitation
wavelengths. The shift in emission maximum as a function
of excitation wavelength is shown in Figure 1B. On
excitation at 280 nm, the fluorescence emission maximum
was at 333 nm. As the excitation wavelength was gradually
shifted toward longer wavelengths, the emission maxima also
showed a concomittant red shift. On excitation at 305 nm,
the emission maximum was obtained at 340 nm. This
corresponds to a REES of 7 nm. Such a red edge excitation
shift was however not visible in the case of the protein that
was denatured with 8 M urea. The observed REES thus
implies that the environment experienced by the tryptophans
in tubulin is motionally restricted, i.e., offers considerable
restriction to the reorientation of the solvent dipoles in the
excited state and is induced by the native conformation of
tubulin. At higher temperatures, the rate of solvent reorientation will be enhanced and emission will be dominated
from the solvent-relaxed states (Lakowicz & KeatingNakamoto, 1984). Thus, the magnitude of the REES is
expected to decrease as the temperature is raised. Results
of such an experiment are shown in Figure 1C. As seen
from the figure, at 37 °C, the extent of the REES is decreased
to 5 nm, as compared to 7 nm at lower temperatures,
indicating that solvent relaxation around the motionally
restricted tryptophans does occur at an enhanced rate at this
temperature.
Fluorescence polarization is also known to show a
dependence on excitation wavelength in viscous solutions
and condensed phases. There are many reports that show
Guha et al.
FIGURE 2: Effect of changing the excitation wavelength on (A)
steady state polarization of tubulin tryptophans in the absence (b)
and presence (O) of 8 M urea, keeping the emission wavelength at
333 nm, and (B) mean fluorescence lifetimes of tubulin, the
emission wavelength being 340 nm. The tubulin concentration was
2 µM, and the temperature was 25 °C.
that fluorescence polarization increases with increasing
excitation wavelength, when the emission wavelength is kept
constant (Weber, 1960a,b; Lynn & Fasman, 1968; Weber
& Shinitzky, 1970; Valeur & Weber, 1977a,b, 1978; Lakowicz, 1983, 1984; Demchenko, 1986; Chattopadhyay &
Mukherjee, 1993). As discussed above, red edge excitation
selects for the solvent-relaxed fluorophores, i.e., those that
interact strongly with the surrounding solvent dipoles. Due
to such strong interactions with the polar solvent molecules
in the excited state, these fluorophores will rotate more
slowly compared to the unrelaxed ones (selectively excited
at lower wavelengths), giving rise to higher polarization at
longer excitation wavelengths (Lakowicz, 1984). Figure 2A
shows the change in fluorescence polarization of the tubulin
tryptophans with increasing excitation wavelength, keeping
the emission wavelength constant at 333 nm. The polarization increases sharply upon red edge excitation with a
characteristic dip at 290 nm (Mukherjee & Chattopadhyay,
1994). Such an increase in polarization occurs to a much
lower extent in the case of the protein treated with 8 M urea.
This is further indicative of the motional restriction experienced by the tryptophans in tubulin.
It is known that tryptophan has two overlapping S0 f S1
electronic transitions (1La and 1Lb) which are almost perpendicular to each other (Weber, 1960a; Song & Kurtin,
1969; Yamamoto & Tanaka, 1972; Eftink, 1991). Both S0
f 1La and S0 f 1Lb transitions occur in the 260-300 nm
range. In nonpolar solvents, 1La has a higher energy than
1L . However, in polar solvents, the energy level of 1L is
b
a
lowered, making it the lowest-energy state. This inversion
is believed to occur because the 1La transition has a higher
dipole moment (as it is directed through the ring NH group)
and can have dipole-dipole interactions with polar solvent
molecules. Whether 1La or 1Lb is the lowest S1 state,
equilibration between these two states is believed to be very
fast (on the order of 10-12 s) so that only emission from the
lower S1 state is observed (Ruggiero et al., 1990). In a
A REES Study of Tubulin
Biochemistry, Vol. 35, No. 41, 1996 13429
Table 1: Lifetimes of Tryptophans in Tubulin as a Function of
Excitation Wavelengtha
excitation
wavelength (nm)
R1
τ1 (ns)
R2
τ2 (ns)
χ2
294
297
302
313
316
0.68
0.78
0.85
0.99
0.98
1.02
0.73
0.68
0.50
0.53
0.32
0.22
0.15
0.01
0.02
4.50
4.51
4.38
2.48
3.06
0.96
1.04
0.93
1.64
1.21
a
Emission wavelength was 340 nm.
motionally restricted polar environment, absorption at the
red edge photoselects the lowest-energy S1 (1La in this case),
and thus, the polarization is high since only depolarization
due to small angular differences between the absorption and
emission transition moments and solvent reorientation, if any,
occurs. Excitation at the shorter wavelengths, however,
populates both 1La and 1Lb states. Equilibration between
these two states produces a depolarization due to the
approximately 90° angular difference between 1La and 1Lb
moments. Thus, near 290 nm, there is a dip in polarization
due to maximal absorption by the 1Lb state. Figure 2A shows
such a characteristic dip around 290 nm in the excitation
polarization spectrum of tubulin. Thus, the sharp increase
in polarization toward the red edge of the absorption band
is probably because the extent of depolarization in tubulin
is reduced at the red edge not only due to the decreased
rotational rate of the fluorophore in a solvent-relaxed state
but also due to photoselection of predominantly 1La transition,
which in turn reduces the contribution to depolarization
because of 1Lb f 1La equilibration.
In addition to steady state polarization, fluorescence
lifetimes are also known to change with excitation wavelengths in motionally restricted media (Castelli & Forster,
1973; Conti & Forster, 1974; Valeur & Weber, 1978;
Demchenko, 1985; Mukherjee & Chattopadhyay, 1994).
When a fluorophore in a restricted environment is excited
at its mean excitation wavelength, a majority of fluorophores
will emit at the steady state fluorescence emission maximum
for that particular excitation wavelength. Also, a majority
of fluorophores will have a mean lifetime associated with
them, if the decay of fluorescence intensity is monitored at
these mean excitation and emission wavelengths. However,
for lifetime measurements upon red edge excitation, keeping
the emission wavelength (although it is now no longer the
emission maximum) unchanged, preselection of only that
small population of fluorophores will occur which have
emitted early, i.e., around which solvent relaxation has not
yet taken place and shifted their emission toward the mean,
red-shifted emission maximum for that excitation wavelength. Thus, fluorescence lifetimes decrease across the
excitation spectrum of a fluorophore under conditions where
it shows a REES. Table 1 shows the lifetimes of the
tryptophan residues in tubulin as a function of excitation
wavelength, keeping the emission wavelength constant at 340
nm. The emission wavelength was fixed at 340 nm in order
to eliminate interference due to scattering at longer excitation
wavelengths. The fluorescence decay of the tryptophans of
tubulin could be fitted to a biexponential function at all
excitation wavelengths, with a short lifetime component, the
preexponential factor associated with which increased at the
red edge of excitation; and a relatively longer lifetime
component, the preexponential factor for which decreased
FIGURE 3: Effect of changing the emission wavelength on (A) mean
fluorescence lifetimes of tubulin tryptophans upon excitation at 297
nm and (B) fluorescence polarization in the absence (b) and
presence (O) of 8 M urea, keeping the excitation wavelength
constant at 280 nm. The tubulin concentration was 2 µM, and the
temperature was 25 °C.
Table 2: Lifetimes of Tryptophans in Tubulin as a Function of
Emission Wavelengtha
emission
wavelength (nm)
R1
τ1 (ns)
R2
τ2 (ns)
χ2
330
340
350
360
370
380
0.22
0.22
0.39
0.35
0.34
0.58
4.82
4.51
4.81
5.81
6.34
2.62
0.78
0.78
0.61
0.65
0.66
0.42
1.11
0.73
1.25
2.99
3.24
6.10
1.30
1.04
0.99
1.02
0.99
0.94
a
Excitation wavelength was 297 nm.
concomitantly. Mean lifetimes were calculated according
to eq 3 and plotted as a function of excitation wavelength in
Figure 2B. As seen from the figure, mean lifetimes of
tubulin decreased steadily (77%) as the excitation wavelength
was increased from 294 to 316 nm.
On the other hand, fluorescence lifetimes for a polar
fluorophore in viscous medium are often found to increase
with increasing emission wavelength when the excitation
wavelength is kept constant (Easter et al., 1976, 1978;
Lakowicz & Cherek, 1980; Matayoshi & Kleinfeld, 1981;
Lakowicz et al., 1983a,b; Demchenko & Shcherbatska, 1985;
Mukherjee & Chattopadhyay, 1994). Figure 3A shows the
change in the mean lifetime of tubulin with the change in
emission wavelength, keeping the excitation wavelength
constant at 297 nm. At the emission wavelength was
gradually increased from 330 to 380 nm, the mean lifetime
of tubulin increased by 52%. The decay of fluorescence
intensity at all emission wavelengths was fitted to a biexponential function, and the preexponential factors associated
with the two lifetime components are shown in Table 2. Such
increasing lifetimes across the emission spectrum of a
fluorophore may be interpreted as follows. Shorter wavelengths of emission select predominantly for unrelaxed
fluorophores which are decaying rapidly both at the rate of
fluorescence emission at that excitation wavelength and also
at the rate of solvent reorientation to longer emission
wavelengths. Hence, observed lifetimes at these emission
13430 Biochemistry, Vol. 35, No. 41, 1996
Guha et al.
FIGURE 4: Fluorescence emission maxima of tryptophans as a
function of excitation wavelength in free tubulin (O) and in the
tubulin-colchicine complex (b). The tubulin concentration was
5 µM, and it was incubated with 10 µM colchicine at 37 °C for 1
h to obtain the tubulin-colchicine complex. Fluorescence emission
maxima were monitored at 25 °C.
wavelengths will be shorter compared to that obtained at the
red edge of emission. Here, solvent-relaxed fluorophores
are selected preferentially which have spent enough time in
the excited state to allow increasingly larger extents of dipole
reorientation around them; i.e., lifetimes are longer.
It follows from the above argument that this subclass of
solvent-relaxed fluorophores, having a longer excited state
lifetime compared to that of the unrelaxed ones, should thus
have more time to rotate in the excited state; i.e., polarization
should decrease across the emission spectrum. It has been
seen that fluorescence polarization decreases across the
emission spectrum of a fluorophore when it is excited at its
mean excitation wavelength (Matayoshi & Kleinfeld, 1981;
Lakowicz et al., 1983b; Demchenko & Shcherbatska, 1985;
Sommer et al., 1990; Chattopadhyay & Mukherjee, 1993).
Figure 3B shows the variation in polarization of tryptophans
as a function of emission wavelength on excitation of tubulin
at 280 nm. While the polarization of the tubulin tryptophans
decreases steadily across the emission spectrum, that of the
tryptophans in unfolded tubulin remains practically invariant.
The REES studies (both steady state and time-resolved)
reported above thus prove that the tryptophan residues in
tubulin are present in a viscous environment where mobility
is restricted and solvent relaxation dynamics occurs on the
nanosecond time scale or longer. Tubulin being a multitryptophan protein, our data are indicative of the average
environment to which the eight tryptophans of tubulin are
exposed and it can only be said that there is at least one
tryptophan which is sufficiently restricted for the REES of
fluorescence to occur. However, it is difficult to exactly
localize the tryptophan(s) responsible for the REES by
spectroscopic studies alone.
In an attempt to identify which of the eight tryptophans
of tubulin are the major contributors to the REES effect
reported above, we carried out REES studies of the tryptophans in the colchicine-tubulin complex. Figure 4 shows
a plot of the emission maxima of the tryptophans in the
tubulin-colchicine complex and also in free tubulin with
changing excitation wavelength. As reported above, in
tubulin, the emission maximum shifts by about 7 nm (from
333 to 340 nm) upon changing excitation wavelength from
280 to 305 nm. We found that the fluorescence emission
maximum of the tubulin-colchicine complex is at 338 nm
FIGURE 5: Red edge excitation shifts of drug fluorescence in
complexes of tubulin with colchicine (b), deacetylcolchicine (4),
deacetamidocolchicine (2), and AC (O). Tubulin and drug
concentrations were 5 and 10 µM, respectively, and the temperature
was 25 °C. Structures of AC compound, colchicine, and its B-ring
analogs used in this study are also shown in the figure.
when it is excited at 280 nm. Changing the excitation
wavelength to 305 nm causes a shift in the emission
maximum to 344 nm, corresponding to a REES of 6 nm.
Thus, although the REES in the tubulin-colchicine complex
occurs to almost the same extent as in tubulin, the emission
maximum values at each excitation wavelength are redshifted in the former compared to those in the latter. Now,
it is known that the colchicine binding site on tubulin is in
close proximity to one or more tryptophan(s) and that energy
transfer, from the tryptophan(s) to bound colchicine, occurs
(Garland, 1978; Andreu & Timasheff, 1982). Such energy
transfer to colchicine from one or more tryptophan(s) near
its binding site will make these tryptophans optically silent
as far as emission is concerned. These tryptophans are
probably present in a very hydrophobic environment, i.e.,
emit at a lower wavelength compared to more exposed ones,
as shutting off of fluorescence emission from them causes
an overall red shift in the emission maximum of tubulin.
Since a polar environment is a primary criterion for the REES
effect to take place, it is expected that the tryptophan(s)
present near the colchicine binding site on tubulin and
involved in energy transfer with bound colchicine will not
contribute significantly to the observed REES.
Red Edge Excitation of Colchicine Complexed with Tubulin. The REES of colchicine fluorescence also was
observed in colchicine-tubulin complexes. A plot of
emission maxima of colchicine against different excitation
wavelengths is given in Figure 5. When the colchicinetubulin complex was excited at 350 nm, which is the
wavelength central to the absorption band of colchicine, the
emission maximum was at 433 nm. The emission maximum
got progressively red-shifted upon red edge excitation until
it was at 440 nm on excitation at 400 nm, corresponding to
a REES of 7 nm. We have stated above that the tryptophan(s) of tubulin, which are very near to the colchicine binding
site and participate in energy transfer with bound colchicine,
A REES Study of Tubulin
are exposed to a nonpolar environment. However, the REES
of colchicine fluorescence in the tubulin-colchicine complex
indicates that, unlike the environment of these tryptophan(s), the environment of bound colchicine is significantly polar
and viscous.
An interesting observation was noted when REES studies
were carried out in complexes of tubulin with some B-ring
analogs of colchicine, viz. 2-methoxy-5-(2′,3′,4′-trimethoxyphenyl)tropone (AC), deacetamidocolchicine (DAAC), and
deacetylcolchicine (structures shown in Figure 5). The REES
of fluorescence emission maxima was totally absent when
both AC (a biphenyl analog of colchicine that lacks the B
ring) and DAAC (which lacks the side chain at the C-7
position of the B ring) bound to tubulin were excited at the
red edge of their absorption bands (Figure 5). On the other
hand, the fluorescence of deacetylcolchicine (having an
amino substituent at the C-7 position of the B ring) bound
to tubulin showed a REES; the emission maximum shifted
from 432 to 436 nm as the excitation wavelength was
increased from 350 to 400 nm.
In order to confirm the observations reported above, we
carried out lifetime measurements of colchicine when bound
to tubulin, at different excitation and emission wavelengths.
The fluorescence lifetime of colchicine in its complex with
tubulin was previously determined by phase fluorimetry and
found to be 1.14 ns ((0.02), at an ionic strength of 0.1 M,
excitation and emission wavelengths being 353 and 430 nm,
respectively (Ide & Engelborghs, 1981). The decay of
fluorescence intensity of colchicine was fitted to a biexponential function at all excitation and emission wavelengths
studied. A decay profile with biexponential fitting and the
various statistical parameters used to check the goodness of
fit are shown in Figure 6A. Figure 6B shows the variation
in mean lifetime of colchicine as a function of excitation
wavelength. As the excitation wavelength was increased
from 354 nm, the mean lifetime decreased gradually until it
was almost 44% decreased when the excitation wavelength
was 390 nm. The emission wavelength was kept constant
at 430 nm for these measurements. The mean lifetime of
colchicine was also measured at different emission wavelengths, keeping the excitation wavelength fixed at 358 nm.
As seen from Figure 6C, the lifetime of the excited state
fluorophore increased by about 72% with increasing emission
wavelength from 430 to 500 nm. Thus, colchicine lifetimes
decrease across its excitation spectrum and increase across
its emission spectrum as expected in the case of a polar
fluorophore in motionally restricted media. Such dependence
of fluorescence lifetimes on excitation and emission wavelengths occurred to a significantly lesser extent in the case
of DAAC (Figure 6B,C).
Our lifetime studies with colchicine thus support the steady
state data and confirm that the colchicine binding site on
tubulin is indeed polar and viscous where solvent relaxation
around the excited state molecule is considerably restricted.
Also, the REES of colchicine analogs appears to be
significantly influenced by heterogeneity in ligand structure
as proven by the fact that some structural analogs of
colchicine, binding at the same site on tubulin, did not show
a REES like colchicine.
DISCUSSION
Red edge excitation shifts of fluorescence emission
maxima have often been used to study the organization and
Biochemistry, Vol. 35, No. 41, 1996 13431
FIGURE 6: (A) Time-resolved decay of fluorescence intensity of
colchicine bound to tubulin. Excitation and emission wavelengths
were 355 and 430 nm, respectively. The peak on the left is the
lamp profile, while that on the right represents the decay profile
fitted to a biexponential function. The two lower plots show the
weighted residuals and the autocorrelation function of the weighted
residuals. (B) Changes in mean fluorescence lifetimes of colchicine
(O) and DAAC (b), bound to tubulin, with changing excitation
wavelength, the emission wavelength being 420 nm. (C) Mean
fluorescence lifetimes of colchicine (O) and DAAC (b), bound to
tubulin, as a function of emission wavelength, keeping the excitation
wavelength constant at 358 nm. Tubulin and drug concentrations
were 5 and 10 µM, respectively, in all cases. All data were taken
at 25 °C.
dynamics of biological systems such as membranes and
proteins. The necessary conditions for giving rise to a REES
can be summarized as follows. (i) The fluorophore should
13432 Biochemistry, Vol. 35, No. 41, 1996
be polar. (ii) The surrounding solvent should be polar. (iii)
Upon excitation, the dipole moment of the fluorophore should
change (Mukherjee et al., 1994). (iv) The solvent relaxation
process around the excited state dipole should be comparable
to or slower than the fluorescence lifetime; i.e., the environment should be motionally restricted.
Our results cited above prove that, among the eight
tryptophans of tubulin, there is at least one present in a
viscous environment that restricts the solvent relaxation
process around them in the excited state. Although we were
unable to localize the tryptophan(s) responsible for the REES
effect, our results indicate that the tryptophan(s) present near
the colchicine binding site on tubulin and involved in energy
transfer with bound colchicine probably does not contribute
to the REES. Also, the environment to which these
tryptophans are exposed appears to be extremely hydrophobic.
Although, colchicine bound to tubulin participates in
energy transfer with these tryptophan(s) present in a highly
hydrophobic region of tubulin, the environment around
colchicine was found to be sufficiently polar and viscous
for a REES to occur. It has been reported previously that
immobilization of colchicine occurs at its binding site on
tubulin (Bhattacharyya & Wolff, 1984). Surprisingly, we
found that fluorescence of the colchicine analogs, AC and
DAAC, did not show any REES, while that of deacetylcolchicine shows a REES to a lesser extent compared to
colchicine, although these drugs have the same binding site
on tubulin. One possible reason for such a marked difference
in behavior can be the structural differences (B-ring side
chain) between the analogs, which may lead to differences
in the dipole moment of these molecules in the excited state,
which in turn will affect the REES. It is known that the B
ring, or more specifically, the substituent at its C-7 position
plays an important role in regulating the binding reaction
between colchicine and tubulin (Bhattacharyya et al., 1986).
The kinetic and thermodynamic parameters of the binding
reaction have been found to be strictly controlled at different
points of the analog structure. AC binding to tubulin is
extremely rapid, highly reversible (Ray et al., 1981), and
enthalpy-driven (Bane et al., 1984). Introduction of the B
ring (DAAC) and inclusion of an increasingly bulky substituent at the C-7 position of the B ring [deacetylcolchicine
(NH2-DAAC), demecolcine (NHMe-DAAC), N-methyldemecolcine (NMe2-DAAC), and colchicine] results in progressive lowering of on rate and off rate constants of binding
and increasing of activation energy and also converts an
enthalpy-driven reaction to an entropy-driven one (Chakrabarti et al., 1996).
These observations can also be explained on the basis of
a simple and widely accepted model for the colchicinetubulin complex. The colchicine site on tubulin is composed
of two chemically different and independent subsites on the
β-subunit of the protein: one for the A ring of colchicine
and another that binds the ring C (Andreu et al., 1991;
Shearwin & Timasheff, 1994). Ring B makes no contribution to the binding other than regulating the binding
parameters of the reaction. It is generally believed now that
the B-ring substituent of colchicine and its analogs reside
outside the colchicine binding site, facing R-tubulin and also
possibly making contact with it (Pyles & Bane Hastie, 1993;
Wolff & Knipling, 1995). It is probable that such a contact
of the C-7 substituent with the protein would restrict the
Guha et al.
flexibility of the bound drug and the environment around it
at the binding site, a condition which facilitates the REES.
Hence, in colchicine analogs, where there is no such
substituent to make contact with the protein, no REES is
observed. Our studies thus serve as further evidence for the
proposed model of the colchicine-tubulin complex.
Lastly, we say that the REES phenomenon reported here
can give rise to complications in spectroscopic experiments
involving tubulin. In such experiments, excitation of a
fluorophore is often done at the red edge of its absorption
band instead of the absorption maximum (e.g., to selectively
excite tryptophans or minimize inner filter effects). However, under conditions which facilitate a REES, such red edge
excitation will induce a red shift in the emission maximum
and a decrease in the mean lifetime, leading to the incorrect
assumption of the fluorophore being more exposed to the
solvent than it really is. In such cases, fluorescence
characteristics should be interpreted with caution.
ACKNOWLEDGMENT
We thank Prof. D. Balasubramaniam and Dr. D. Chatterji,
Centre for Cellular and Molecular Biology, Hyderabad, for
kindly permitting S.G. to perform the lifetime measurements
on the Photon Technology International LS-100 luminescence spectrophotometer at CCMB.
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